Methods of modulating cytokine activity; related reagents

ABSTRACT

Provided are methods of modulating cytokine activity, e.g., for the purpose of treating immune and inflammatory disorders, including tumors and cancer. Also provided are methods of administering agonists or antagonists of IL-33 and IL-33 receptor.

This application is a Divisional application of co-pending U.S. patentapplication Ser. No. 14/171,629, filed Feb. 3, 2014, which is aContinuation of U.S. Ser. No. 12/239,689, filed Sep. 26, 2008, which isa Continuation of U.S. patent application Ser. No. 11/682,224, filedMar. 5, 2007, which is a Continuation of U.S. patent application Ser.No. 11/059,117, filed Feb. 15, 2005, which claims benefit of U.S.Provisional Patent Application No. 60/545,730, filed Feb. 17, 2004, eachof which is incorporated herein by reference.

The Sequence Listing filed electronically herewith is also herebyincorporated by reference in its entirety (File Name:DX06173USDIV-SEQLIST-20DEC2017.txt; Date Created: Dec. 20, 2017; FileSize: 32 KB.)

FIELD OF THE INVENTION

The present invention relates generally to uses of mammalian cytokines.More specifically, the invention discloses methods of using IL-33, and areceptor for IL-33.

BACKGROUND OF THE INVENTION

The immune system protects individuals from infective agents, e.g.,bacteria, multi-cellular organisms, as well as cancers. This systemincludes several types of lymphoid and myeloid cells such as monocytes,macrophages, dendritic cells (DCs), eosinophils, T cells, B cells, andneutrophils. These lymphoid and myeloid cells often produce signalingproteins known as cytokines. Immune response includes inflammation,i.e., the accumulation of immune cells systemically or in a particularlocation of the body. In response to an infective agent or foreignsubstance, immune cells secrete cytokines which, in turn, modulateimmune cell proliferation, development, differentiation, or migration.Immune response sometimes results in pathological consequences, that is,inflammatory disorders. These inflammatory disorders, which involveimmune cells and cytokines, include, e.g., psoriasis, rheumatoidarthritis, Crohn's disease, multiple sclerosis, and atherosclerosis(see, e.g., Abbas, et al. (eds.) (2000) Cellular and MolecularImmunology, W.B. Saunders Co., Philadelphia, Pa.; Oppenheim and Feldmann(eds.) (2001) Cytokine Reference, Academic Press, San Diego, Calif.;Kaufmann, et al. (2001) Immunobiol. 204:603-613; Saurez andSchultz-Cheery (2000) Dev. Comp. Immunol. 24:269-283; van Reeth andNauwynck (2000) Vet. Res. 31:187-213; Garcia-Sastre (2001) Virology279:375-384; Katze, et al. (2002) Nat. Rev. Immunol. 2:675-687; vanReeth (2000) Vet. Microbiol. 74:109-116; Tripp (2003) Curr. Pharm. Des.9:51-59).

The interleukin-1 (IL-1) family of cytokines contributes to thepathology of inflammatory disorders and proliferative conditions, e.g.,arthritis and cancer. Cytokines of the IL-1 family include IL-1alpha,IL-1beta, IL-1delta, IL-1epsilon, basic fibroblast growth factor, IL-18,CREG and CREG2. IL-1alpha and IL-1beta are biosynthesized as 31 kDapolypeptides that are further processed to mature 17 kDa forms, whileIL-1delta and IL-1epsilon appear not to possess a distinct pro-form(see, e.g., Debets, et al. (2001) J. Immunol. 167:1440-1446; McMahon, etal. (1997) J. Biol. Chem. 272:28202-28205; Irikura, et al. (2002) NewEngl. J. Med. 169:393-398; Kim, et al. (2002) J. Biol. Chem.277:10998-11003).

The IL-1 family also includes IL-1 receptors, i.e., IL-1RI, IL-1RII, andIL-1R accessory protein (a.k.a. IL-1R1, IL-1R2, and IL-1R3,respectively). IL-1alpha and IL-1beta trigger cell signaling by bindingto IL-1R1, while IL-1RII can function as a molecule that absorbscirculating ligand. IL-1 receptor antagonist (IL-1Ra), another IL-1family protein, binds to IL-1 receptor without transmitting a signal andserves as an inhibitor of IL-1. IL-1ra and IL-1delta play similar rolesin antagonizing signaling through receptors, i.e., IL-1ra antagonizesIL-1alpha-mediated signaling via IL-1R1, while IL-1delta antagonizesIL-1epsilon-mediated signaling via IL-1R6 (see, e.g., You, et al. (2001)New Engl. J. Med. 193:101-109). Debets, et al. (2001) J. Immunol.167:1440-1446; Apte and Voronov (2002) Sem. Cancer Biol. 12:277-290;Wong, et al. (1997) Proc. Natl. Acad. Sci. USA 94:227-232).

IL-1 family members play a role in inflammatory conditions, e.g.,rheumatoid arthritis, psoriasis, asthma, chronic obstructive pulmonarydisorder (COPD), sepsis, and inflammatory bowel disorder (IBD).Rheumatoid arthritis (RA) is a common chronic inflammatory disordercharacterized by degradation of joints, e.g., the synovial membrane,cartilage, and bone. The disorder strikes about 1% of the population andcannot be cured. IL-1 stimulates a number of cells involved in arthriticinflammation, e.g., fibroblasts, osteoclasts, chondrocytes, andneutrophils, which may show abnormal proliferation and release enzymescausing joint destruction (see, e.g., (Debets, et al. (1997) J. Immunol.158:2955-2963; Lacey, et al. (2003) Arthritis Rheum. 48: 103-109; Chung(2001) Eur. Resp. J. Suppl. 34: 50s-59s; Freeman and Buchman (2001)Expert Opin. Biol. Ther. 1:301-308; Dinarello (2000) Chest 118:503-508).Krause, et al. (2002) J. Immunol. 169:6610-6616; Choy and Panayi (2001)New Engl. J. Med. 344:907-916; Woolley (2003) New Engl. J. Med.348:1709-1711; Williams, et al. (2000) New Engl. J. Med. 164: 7240-7245;Feldmann and Maini (2001) Annu. Rev. Immunol. 19:163-196; Lacey, et al.,supra; Niki, et al. (2001) J. Clin. Invest. 107:1127-1135; Attur, et al.(2000) J. Biol. Chem. 51:40307-40315).

Proliferative disorders are the second most common cause of death in theUnited States (Anderson (2002) National Vital Statistics Reports50:1-86; Toribara and Sleisenger (2003) New Engl. J. Med. 332:861-867;Janne and Mayer (2000) New Engl. J. Med. 342:1960-1968; Fuchs and Mayer(1995) New Engl. J. Med. 333:32-41). Cytokines of the IL-1 family havebeen implicated in the control and pathology of proliferative disorders,i.e., cancer. IL-1 modulates progression through the cell cycle, e.g.,by changing expression of cyclin-dependent kinases and cyclin-dependentkinase inhibitors. High doses of IL-1beta promote tumor invasiveness,while low doses can promote immune eradication of tumors (see, e.g.,Zeisler, et al. (1998) Eur. J. Cancer 34:931-933; Yoshida, et al. (2002)Brit. J. Cancer 86:1396-1400; Nesbit, et al. (1999) Oncogene18:6469-6476; Dinarello, et al. (1998) J. Leuko. Biol. 63:658-664; Apteand Voronov, supra; Saijo, et al. (2002) New Engl. J. Med. 169: 469-475;Murai, et al. (2001) J. Biol. Chem. 276:6797-6806; Koudssi, et al.(1998) J. Biol. Chem. 273: 25796-25803; Zeki, et al. (1999) J.Endocrinol. 160:67-73; Osawa, et al. (2000) J. Biochem. 127:883-893).

There is an unmet need to treat inflammatory and immune disorders. Thepresent invention fulfils this need by providing methods of usingagonists and antagonists of IL-33 or IL-33 receptor.

SUMMARY OF THE INVENTION

The present invention is based, in part, upon the discovery that anagonist or antagonist of IL-33 or IL-33 receptor (previously known asIL-100 and IL-100 receptor) modulates response to a number of immune andinflammatory conditions.

The present invention provides a method of modulating an immune disorderor condition, comprising administering an effective amount of an agonistor antagonist of IL-33 or IL-33R complex. Also provided is the abovemethod wherein the disorder or condition comprises: a) innate response;b) asthma or allergy; c) multiple sclerosis; d) an inflammatory boweldisorder; e) arthritis; f) infection; g) a cancer or tumor. Furtherprovided is the above method wherein the infection comprises: a) anintracellular pathogen; b) a bacterium; c) a parasite; or d) a virus;and the above method wherein the intracellular pathogen is: a)Leishmania sp.; b) Mycobacterium sp.; c) Listeria sp.; d) Toxoplasmasp.; e) Schistosoma; or f) a respiratory virus. Moreover, the presentinvention provides the above method wherein the immune disorder orconditions comprises TH1-type response or TH2-type response; and theabove method wherein the TH2-type response comprises an early event inTH2-type response; as well as the above method wherein the arthritiscomprises rheumatoid arthritis; osteoarthritis; or psoriatic arthritis.

In another embodiment, the present invention provides the above methodwherein the agonist comprises IL-33 or a nucleic acid; as well as theabove method wherein the nucleic acid encodes IL-33; and the abovemethod wherein the antagonist comprises a binding composition from anantibody that specifically binds IL-33 or a complex of IL-33, T1/ST2 andSIGIRR (IL-33R). In yet another embodiment, the present inventionprovides the above method wherein the binding composition from anantibody comprises a polyclonal antibody; a monoclonal antibody; ahumanized antibody, or a fragment thereof; an Fab, Fv, or F(ab′)₂fragment; a peptide mimetic of an antibody; or a detectable label. Alsoprovided is the above method, wherein wherein the antagonist comprises:a) a soluble IL-33R; b) a small molecule; or c) a nucleic acid; and theabove method wherein the nucleic acid specifically hybridizes with apolynucleotide encoding IL-33; as well as the above method wherein thenucleic acid comprises anti-sense nucleic acid or small interference RNA(siRNA).

In another aspect, the present invention provides a method of modulatingblood cell counts comprising administering an effective amount of anagonist or antagonist of IL-33; and the above method wherein the IL-33agonist increases the counts of total white blood cells; neutrophils;lymphocytes; or eosinophils; as well as the above method wherein theIL-33 antagonist increases the count of platelets; and the above methodwherein the IL-33 antagonist decreases the counts of total white bloodcells; neutrophils; lymphocytes; or eosinophils.

Yet another aspect of the present invention provides a method ofdiagnosing the immune condition or disorder noted above, comprisingcontacting a binding composition to a biological sample, wherein thebinding composition specifically binds to IL-33, and measuring ordetermining the specific binding of the binding composition to thebiological sample. Also provided is a kit for the diagnosis of theimmune condition or disorder of Claim 1, comprising a compartment and abinding composition that specifically binds to: IL-33; an IL-33Rcomplex; a complex of IL-33 and IL-33R; or a nucleic acid encodingIL-33.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows IL-5 production in IL-33+anti-IL-33 antibody treated miceversus IL-33 alone and isotype control antibody treated mice.

FIG. 2 shows CIA disease scores for anti-IL-33 and isotype controltreated mice.

FIG. 3 shows the incidence of CIA in anti-IL-33 and isotype controltreated mice.

FIG. 4 shows the mean number of arthritic paws in mice treated withanti-IL-33 antibody or isotype control antibody.

FIG. 5 shows the EAE disease scores of anti-IL-33 and isotype controltreated mice.

FIG. 6 shows the incidence of EAE in anti-IL-33 and isotype controltreated mice.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As used herein, including the appended claims, the singular forms ofwords such as “a,” “an,” and “the,” include their corresponding pluralreferences unless the context clearly dictates otherwise.

All references cited herein are incorporated herein by reference to thesame extent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference.

I. Definitions

“Activation,” “stimulation,” and “treatment,” as it applies to cells orto receptors, may have the same meaning, e.g., activation, stimulation,or treatment of a cell or receptor with a ligand, unless indicatedotherwise by the context or explicitly. “Ligand” encompasses natural andsynthetic ligands, e.g., cytokines, cytokine variants, analogues,muteins, and binding compositions derived from antibodies. “Ligand” alsoencompasses small molecules, e.g., peptide mimetics of cytokines andpeptide mimetics of antibodies. “Activation” can refer to cellactivation as regulated by internal mechanisms as well as by external orenvironmental factors. “Response,” e.g., of a cell, tissue, organ, ororganism, encompasses a change in biochemical or physiological behavior,e.g., concentration, density, adhesion, or migration within a biologicalcompartment, rate of gene expression, or state of differentiation, wherethe change is correlated with activation, stimulation, or treatment, orwith internal mechanisms such as genetic programming.

“Activity” of a molecule may describe or refer to the binding of themolecule to a ligand or to a receptor, to catalytic activity; to theability to stimulate gene expression or cell signaling, differentiation,or maturation; to antigenic activity, to the modulation of activities ofother molecules, and the like. “Activity” of a molecule may also referto activity in modulating or maintaining cell-to-cell interactions,e.g., adhesion, or activity in maintaining a structure of a cell, e.g.,cell membranes or cytoskeleton. “Activity” can also mean specificactivity, e.g., [catalytic activity]/[mg protein], or [immunologicalactivity]/[mg protein], concentration in a biological compartment, orthe like. “Proliferative activity” encompasses an activity thatpromotes, that is necessary for, or that is specifically associatedwith, e.g., normal cell division, as well as cancer, tumors, dysplasia,cell transformation, metastasis, and angiogenesis.

“Administration” and “treatment,” as it applies to the administration ofan agonist or antagonist of IL-33, e.g., to an animal, human,experimental subject, cell, tissue, organ, or biological fluid, refersto contact of an exogenous pharmaceutical, therapeutic, diagnosticagent, compound, or composition to the animal, human, subject, cell,tissue, organ, or biological fluid. “Administration” and “treatment” canrefer, e.g., to therapeutic, placebo, pharmacokinetic, diagnostic,research, and experimental methods. “Treatment of a cell” encompassescontact of a reagent to the cell, as well as contact of a reagent to afluid, where the fluid is in contact with the cell. “Administration” and“treatment” also means in vitro and ex vivo treatments, e.g., of a cell,by a reagent, diagnostic, binding composition, or by another cell.“Treatment,” as it applies to a human, veterinary, or research subject,refers to therapeutic treatment, prophylactic or preventative measures,to research and diagnostic applications. “Treatment” as it applies to ahuman, veterinary, or research subject, or cell, tissue, or organ,encompasses contact of an IL-33 agonist or IL-33 antagonist to a humanor animal subject, a cell, tissue, physiological compartment, orphysiological fluid. “Treatment of a cell” also encompasses situationswhere the IL-33 agonist or IL-33 antagonist contacts IL-33 receptor(T1/ST2), e.g., in the fluid phase or colloidal phase, as well assituations where the agonist or antagonist contacts a fluid, e.g., wherethe fluid is in contact with a cell or receptor, but where it has notbeen demonstrated that the agonist or antagonist contacts the cell orreceptor.

“Binding composition” refers to a molecule, small molecule,macromolecule, antibody, a fragment or analogue thereof, or solublereceptor, capable of binding to a target, where the target is, e.g.,IL-33 or IL-33R. “Binding composition” also may refer to a complex ofmolecules, e.g., a non-covalent complex, to an ionized molecule, and toa covalently or non-covalently modified molecule, e.g., modified byphosphorylation, acylation, cross-linking, cyclization, or limitedcleavage, which is capable of binding to a target. “Binding composition”may also refer to a molecule in combination with a stabilizer,excipient, salt, buffer, solvent, or additive, capable of binding to atarget. “Binding” may be defined as an association of the bindingcomposition with a target where the association results in reduction inthe normal Brownian motion of the binding composition, in cases wherethe binding composition can be dissolved or suspended in solution.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences or, where the nucleic acid does not encode an amino acidsequence, to essentially identical nucleic acid sequences. Because ofthe degeneracy of the genetic code, a large number of functionallyidentical nucleic acids may encode any given protein. As to amino acidsequences, one of skill will recognize that an individual substitutionto a nucleic acid, peptide, polypeptide, or protein sequence whichsubstitutes an amino acid or a small percentage of amino acids in theencoded sequence for a conserved amino acid is a “conservativelymodified variant.” Conservative substitution tables providingfunctionally similar amino acids are well known in the art. An exampleof a conservative substitution is the exchange of an amino acid in oneof the following groups for another amino acid of the same group (U.S.Pat. No. 5,767,063 issued to Lee, et al.; Kyte and Doolittle (1982) J.Mol. Biol. 157: 105-132):

(1) Hydrophobic: Norleucine, Ile, Val, Leu, Phe, Cys, or Met;

(2) Neutral hydrophilic: Cys, Ser, Thr;

(3) Acidic: Asp, Glu; (4) Basic: Asn, Gln, His, Lys, Arg;

(5) Residues that influence chain orientation: Gly, Pro;

(6) Aromatic: Trp, Tyr, Phe;

(7) Small amino acids: Gly, Ala, Ser.

“Derived” can be used to describe, e.g., deriving the structure of apeptide, oligopeptide, or polypeptide from a parent peptide,oligopeptide, or polypeptide, such as an antibody. In this context,derived encompasses, e.g., peptide structures where the peptide has thesame sequence as a sequence found within the parent, e.g., where thepeptide is identical to the parent but with a truncation at theN-terminus, C-terminus, or both N- and C-termini of the parent, or witha truncation and a fusion, or with a fusion only. Derived also meansthat the peptide has the same sequence as found in the parent, but withconservative amino acid changes, or with deletions or insertions, wherethe deletions or insertions preserve a biological property in thepeptide that is inherent in the parent. “Derived” encompasses situationswhere the peptide or polypeptide is synthesized using the parent as astarting compound, and where the peptide or polypeptide is synthesizedde novo, using the structure of the parent as a guide.

“Effective amount” or “therapeutically effective amount,” of the agonistor antagonist of the IL-33 of the present invention, means an amountsufficient to ameliorate a symptom or sign of a disorder orphysiological condition or an amount sufficient to permit or facilitatea diagnosis of the disorder or physiological condition. An effectiveamount for a particular patient or veterinary subject may vary dependingon factors such as the condition being treated, the overall health ofthe patient, the method route and dose of administration and theseverity of side affects (see, e.g., U.S. Pat. No. 5,888,530 issued toNetti, et al.). An effective amount can be the maximal dose or dosingprotocol that avoids significant side effects or toxic effects. Theeffect will result in an improvement of a diagnostic measure, parameter,or detectable signal by at least 5%, usually by at least 10%, moreusually at least 20%, most usually at least 30%, preferably at least40%, more preferably at least 50%, most preferably at least 60%, ideallyat least 70%, more ideally at least 80%, and most ideally at least 90%,where 100% is defined as the diagnostic parameter shown by a normalsubject (see, e.g., Maynard, et al. (1996) A Handbook of SOPs for GoodClinical Practice, Interpharm Press, Boca Raton, Fla.; Dent (2001) GoodLaboratory and Good Clinical Practice, Urch Publ., London, UK).

“Exogenous” refers to substances that are produced outside an organism,cell, or human body, depending on the context. “Endogenous” refers tosubstances that are produced within a cell, organism, or human body,depending on the context.

“Disorder” refers to a pathological state, or a condition that iscorrelated with or predisposes to a pathological state. “Infectiousdisorder” refers, e.g., to a disorder resulting from a microbe,bacterium, parasite, virus, and the like, as well as to aninappropriate, ineffective, or pathological immune response to thedisorder. “Oncogenic disorder” encompasses a cancer, transformed cell,tumor, displasia, angiogenesis, metastasis, and the like, as well as toan inappropriate, ineffective, or pathological immune response to thedisorder.

“Effective amount” means, e.g., an amount of an IL-33 agonist, IL-33antagonist, binding compound or binding composition, sufficient toameliorate a symptom or sign of a disorder, condition, or pathologicalstate. “Effective amount” also relates to an amount of an IL-33 agonist,antagonist, or binding compound or composition, sufficient to allow orfacilitate the diagnosis of a symptom or sign of a disorder, condition,or pathological state.

“Inhibitors” and “antagonists” or “activators” and “agonists” refer toinhibitory or activating molecules, respectively, e.g., for theactivation of, e.g., a ligand, receptor, cofactor, a gene, cell, tissue,or organ. A modulator of, e.g., a gene, a receptor, a ligand, or a cell,is a molecule that alters an activity of the gene, receptor, ligand, orcell, where activity can be activated, inhibited, or altered in itsregulatory properties. The modulator may act alone, or it may use acofactor, e.g., a protein, metal ion, or small molecule. Inhibitors arecompounds that decrease, block, prevent, delay activation, inactivate,desensitize, or down regulate, e.g., a gene, protein, ligand, receptor,or cell. Activators are compounds that increase, activate, facilitate,enhance activation, sensitize, or up regulate, e.g., a gene, protein,ligand, receptor, or cell. An inhibitor may also be defined as acomposition that reduces, blocks, or inactivates a constitutiveactivity. An “agonist” is a compound that interacts with a target tocause or promote an increase in the activation of the target. An“antagonist” is a compound that opposes the actions of an agonist. Anantagonist prevents, reduces, inhibits, or neutralizes the activity ofan agonist. An antagonist can also prevent, inhibit, or reduceconstitutive activity of a target, e.g., a target receptor, even wherethere is no identified agonist.

To examine the extent of inhibition, for example, samples or assayscomprising a given, e.g., protein, gene, cell, or organism, are treatedwith a potential activator or inhibitor and are compared to controlsamples without the inhibitor. Control samples, i.e., not treated withantagonist, are assigned a relative activity value of 100%. Inhibitionis achieved when the activity value relative to the control is about 90%or less, typically 85% or less, more typically 80% or less, mosttypically 75% or less, generally 70% or less, more generally 65% orless, most generally 60% or less, typically 55% or less, usually 50% orless, more usually 45% or less, most usually 40% or less, preferably 35%or less, more preferably 30% or less, still more preferably 25% or less,and most preferably less than 25%. Activation is achieved when theactivity value relative to the control is about 110%, generally at least120%, more generally at least 140%, more generally at least 160%, oftenat least 180%, more often at least 2-fold, most often at least 2.5-fold,usually at least 5-fold, more usually at least 10-fold, preferably atleast 20-fold, more preferably at least 40-fold, and most preferablyover 40-fold higher.

Endpoints in activation or inhibition can be monitored as follows.Activation, inhibition, and response to treatment, e.g., of a cell,physiological fluid, tissue, organ, and animal or human subject, can bemonitored by an endpoint. The endpoint may comprise a predeterminedquantity or percentage of, e.g., an indicia of inflammation,oncogenicity, or cell degranulation or secretion, such as the release ofa cytokine, toxic oxygen, or a protease. The endpoint may comprise,e.g., a predetermined quantity of ion flux or transport; cell migration;cell adhesion; cell proliferation; potential for metastasis; celldifferentiation; and change in phenotype, e.g., change in expression ofgene relating to inflammation, apoptosis, transformation, cell cycle, ormetastasis (see, e.g., Knight (2000) Ann. Clin. Lab. Sci. 30:145-158;Hood and Cheresh (2002) Nature Rev. Cancer 2:91-100; Timme, et al.(2003) Curr. Drug Targets 4:251-261; Robbins and Itzkowitz (2002) Med.Clin. North Am. 86:1467-1495; Grady and Markowitz (2002) Annu. Rev.Genomics Hum. Genet. 3:101-128; Bauer, et al. (2001) Glia 36:235-243;Stanimirovic and Satoh (2000) Brain Pathol. 10:113-126).

An endpoint of inhibition is generally 75% of the control or less,preferably 50% of the control or less, more preferably 25% of thecontrol or less, and most preferably 10% of the control or less.Generally, an endpoint of activation is at least 150% the control,preferably at least two times the control, more preferably at least fourtimes the control, and most preferably at least 10 times the control.

“Expression” refers to a measure of mRNA or polypeptide encoded by aspecific gene. Units of expression may be a measure of, e.g., the numberof molecules of mRNA or polypeptide/mg protein, the number of moleculesof mRNA or polypeptide/cell, in measurements of expression by cell,tissue, cell extract, or tissue extract. The units of expression may berelative, e.g., a comparison of signal from control and experimentalmammals or a comparison of signals with a reagent that is specific forthe mRNA or polypeptide versus with a reagent that is non-specific.

“Hybridization” that is specific or selective typically occurs whenthere is at least about 55% homology over a stretch of at least about 30nucleotides, preferably at least about 75% over a stretch of about 25nucleotides, and most preferably at least about 90% over about 20nucleotides (see, e.g., Kanehisa (1984) Nucleic Acids Res. 12:203-213).Hybridization under stringent conditions, e.g., of a first nucleic acidto a second nucleic acid, are those that: (1) Employ low ionic strengthand high temperature for washing, for example, 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) Employ during hybridization a denaturing agent, such as formamide,for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1%Ficoll® (Sigma-Aldrich, St. Louis, Mo.)/0.1% polyvinylpyrrolidone/50 mMsodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mMsodium citrate at 42° C.; (3) Employ 50% formamide, 5×SSC (0.75 M NaCl,0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodiumpyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50ng/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42°C. in 0.2×SSC and 0.1% SDS; or (4) Employ a buffer of 10% dextransulfate, 2×SSC (sodium chloride/sodium citrate), and 50% formamide at55° C., followed by a high-stringency wash consisting of 0.1×SSCcontaining EDTA at 55° C. (U.S. Pat. No. 6,387,657 issued to Botstein,et al.).

Stringent conditions for hybridization of nucleic acids are a functionof salt, temperature, organic solvents, and chaotropic agents. Stringenttemperature conditions will usually include temperatures in excess ofabout 30° C., more usually in excess of about 37° C., typically inexcess of about 45° C., more typically in excess of about 50° C.,preferably in excess of about 65° C., and more preferably in excess ofabout 70° C. Stringent salt conditions will ordinarily be less thanabout 1 M, more ordinarily less than about 500 mM, usually less thanabout 400 mM, more usually less than about 300 mM, typically less thanabout 200 mM, preferably less than about 100 mM, and more preferablyless than about 80 mM, even down to less than about 20 mM. However, thecombination of parameters is more important than the measure of anysingle parameter (Wetmur and Davidson (1968) J. Mol. Biol. 31:349-370).

“Immune condition” or “immune disorder” encompasses, e.g., pathologicalinflammation, an inflammatory disorder, and an autoimmune disorder ordisease. “Immune condition” also refers to infections, persistentinfections, and proliferative conditions, such as cancer, tumors, andangiogenesis, including infections, tumors, and cancers that resistirradication by the immune system. “Cancerous condition” includes, e.g.,cancer, cancer cells, tumors, angiogenesis, and precancerous conditionssuch as dysplasia.

“Inflammatory disorder” means a disorder or pathological condition wherethe pathology results, in whole or in part, from, e.g., a change innumber, change in rate of migration, or change in activation, of cellsof the immune system. Cells of the immune system include, e.g., T cells,B cells, monocytes or macrophages, antigen presenting cells (APCs),dendritic cells, microglia, NK cells, NKT cells, neutrophils,eosinophils, mast cells, or any other cell specifically associated withthe immunology, for example, cytokine-producing endothelial orepithelial cells.

“Inflammatory disorder” means a disorder or pathological condition wherethe pathology results, in whole or in part, from an increase in thenumber and/or increase in activation of cells of the immune system,e.g., of T cells, B cells, monocytes or macrophages, alveolarmacrophages, dendritic cells, NK cells, NKT cells, neutrophils,eosinophils, or mast cells.

“IL-33 Receptor”, “IL-33R”, or “IL-33R complex” as used herein shallmean the association of two IL-1R family members, T1/ST2 and SIGIRR toform receptor complex responsive to stimulation with IL-33.

“Ligand” refers, e.g., to a small molecule, peptide, polypeptide, andmembrane associated or membrane-bound molecule, or complex thereof, thatcan act as an agonist or antagonist of a receptor. “Ligand” alsoencompasses an agent that is not an agonist or antagonist, but that canbind to the receptor without significantly influencing its biologicalproperties, e.g., signaling or adhesion. Moreover, “ligand” includes amembrane-bound ligand that has been changed, e.g., by chemical orrecombinant methods, to a soluble version of the membrane-bound ligand.By convention, where a ligand is membrane-bound on a first cell, thereceptor usually occurs on a second cell. The second cell may have thesame or a different identity as the first cell. A ligand or receptor maybe entirely intracellular, that is, it may reside in the cytosol,nucleus, or some other intracellular compartment. The ligand or receptormay change its location, e.g., from an intracellular compartment to theouter face of the plasma membrane. The complex of a ligand and receptoris termed a “ligand receptor complex.” Where a ligand and receptor areinvolved in a signaling pathway, the ligand occurs at an upstreamposition and the receptor occurs at a downstream position of thesignaling pathway.

A “first polypeptide chain” and a “second polypeptide chain” refers totwo polypeptide chains not linked together by way of a classical peptidebond. Typically, the first polypeptide chain comprises an N-terminus andC-terminus, and the second polypeptide chain comprises anotherN-terminus and another C-terminus, that is, altogether there are twoN-termini and two C-termini. The first polypeptide chain can be encodedby a first vector, while the second polypeptide chain can be encoded bya second vector. The first polypeptide chain and second polypeptidechain can be encoded by one vector, where a first promoter can beoperably linked with the first polypeptide chain and a second promotercan be operably linked with the second polypeptide chain or, in anotherembodiment, expression of both the first and second polypeptide chainscan be operably linked to the same promoter.

“Sensitivity,” e.g., sensitivity of receptor to a ligand, means thatbinding of a ligand to the receptor results in a detectable change inthe receptor, or in events or molecules specifically associated with thereceptor, e.g., conformational change, phosphorylation, nature orquantity of proteins associated with the receptor, or change in geneticexpression mediated by or associated with the receptor.

“Small molecules” are provided for the treatment of physiology anddisorders of tumors and cancers. “Small molecule” is defined as amolecule with a molecular weight that is less than 10 kD, typically lessthan 2 kD, and preferably less than 1 kD. Small molecules include, butare not limited to, inorganic molecules, organic molecules, organicmolecules containing an inorganic component, molecules comprising aradioactive atom, synthetic molecules, peptide mimetics, and antibodymimetics. As a therapeutic, a small molecule may be more permeable tocells, less susceptible to degradation, and less apt to elicit an immuneresponse than large molecules. Small molecules, such as peptide mimeticsof antibodies and cytokines, as well as small molecule toxins aredescribed (see, e.g., Casset, et al. (2003) Biochem. Biophys. Res.Commun. 307:198-205; Muyldermans (2001) J. Biotechnol. 74:277-302; Li(2000) Nat. Biotechnol. 18:1251-1256; Apostolopoulos, et al. (2002)Curr. Med. Chem. 9:411-420; Monfardini, et al. (2002) Curr. Pharm. Des.8:2185-2199; Domingues, et al. (1999) Nat. Struct. Biol. 6:652-656; Satoand Sone (2003) Biochem. J. 371:603-608; U.S. Pat. No. 6,326,482 issuedto Stewart, et al).

“Soluble receptor” refers to receptors that are water-soluble and occur,e.g., in extracellular fluids, intracellular fluids, or weaklyassociated with a membrane. Soluble receptor further refers to receptorsthat are engineered to be water soluble. For T1/ST2, the soluble orextracellular domain is defined as residues 1-337 of SEQ ID NO: 6(human) and residues 1-342 of SEQ ID NO: 8 (mouse). For SIGIRR, thesoluble or extracellular domain is defined as residues 1-118 of SEQ IDNO: 10 (human) and residues 1-117 of SEQ ID NO: 12 (mouse).

“Specificity of binding,” “selectivity of binding,” and the like, referto a binding interaction between a predetermined ligand and apredetermined receptor that enables one to distinguish between thepredetermined ligand and other ligands, or between the predeterminedreceptor and other receptors. “Specifically” or “selectively” binds,when referring to a ligand/receptor, antibody/antigen, or other bindingpair, indicates a binding reaction that is determinative of the presenceof the protein in a heterogeneous population of proteins and otherbiologics. Thus, under designated conditions, a specified ligand bindsto a particular receptor and does not bind in a significant amount toother proteins present in the sample. The antibody, or bindingcomposition derived from the antigen-binding site of an antibody, bindsto its antigen with an affinity that is at least two fold greater,preferably at least ten times greater, more preferably at least 20-timesgreater, and most preferably at least 100-times greater than theaffinity to any other antigen. In a preferred embodiment the antibodywill have an affinity that is greater than about 10⁹ liters/mol (see,e.g., Munsen, et al. (1980) Analyt. Biochem. 107:220-239).

II. General

The present invention provides methods for the modulation or treatmentof a number of immune conditions and disorders. In particular, thepresent invention provides agonists and antagonists of IL-33 for thetreatment and diagnosis of, e.g., asthma, allergies, arthritis, andresponse to intracellular pathogens, such as parasites, and response todisorders involving granulomas, e.g., tuberculosis, sarcoidosis, andCrohn's disease.

Naïve T cells appear not to express T1/ST2 on their surface, whereasexpression is induced after contact with antigens on differentiated TH2effector cells. T1/ST2 has been used as a marker for TH2-type T cells.T1/ST2 is also expressed on mast cells and fibroblasts

Studies with T1/ST2 knockout mice seems to suggest that T1/ST2 does notplay a part in the differentiation of naïve CD4⁺ T cells to TH2-type Tcells, though these results appear to be a function of the nature of theassays used, e.g., which pathogenic organism is used in challengestudies, or which phase of TH2-response is studied. Evidence alsosuggests a role for T1/ST2 in early events in TH2-response (see, e.g.,Kropf, et al. (2002) Infect. Immunity 70:5512-5520; Hoshino, et al.(1999) J. Exp. Med. 190:1541-1547; Senn, et al. (2000) Eur. J. Immunol.30:1929-1938; Townsend, et al. (2000) J. Exp. Med. 191:1069-1075).

Anti-T1/ST2 antibodies have been used in a number of studies addressingthe role of T1/ST2 in immune function, while other studies have examinedT1/ST2 expression animal models for immune response. Treatment withanti-T1/ST2 antibodies resulted in decreased TH2-type immune responses.The antibody inhibited eosinophil infiltration, IL-5 production, andIgE-production. Infections by Schistosoma provoked an up-regulation ofT1/ST2, e.g., as determined by assessing expression in lung and livergranulomas. Animal models for asthma, e.g., treatment with house dustmite extract or with ovalbumin, resulted in increased expression ofT1/ST2 on CD4⁺ T cells, indicating a role for T1/ST2 in allergic orasthmatic responses. Studies with BALB/c mice revealed that treatingwith anti-T1/ST2 antibody induced higher TH1-type response, enhancingthe ability of CD4⁺ T cells to respond to IL-12. Anti-T1/ST2 antibodiesalso reduce lesions due to Leishmania major infections, and reducedexpression of TH2-type cytokines. An animal model of arthritis (collageninduced arthritis; CIA) was exacerbated by anti-T1/ST2 antibodies. Inparticular, T1/ST2 functions in early events in the generation ofTH2-type responses. Chronic exposure to various allergens resulted inincreased expression of T1/ST2 on CD4⁺ T cells. T1/ST2 plays a role inmediating innate response, as anti-T1/ST2 antibodies exacerbate thetoxic effects of lipopolysaccharide (LPS). Antibodies to T1/ST2 alsomodulated immune response to viruses, e.g., respiratory syncytial virus(see, e.g., Xu, et al. (1998) J. Exp. Med. 187:787-794; Lohning, et al.(1998) Proc. Natl. Acad. Sci. USA 95:6930-6935; Coyle, et al. (1999) J.Exp. Med. 190:895-902; Lohning, et al. (1999) J. Immunol. 162:3882-3889;Johnson, et al. (2003) Am. J. Respir. Crit. Care Med. 169:378-385;Kropf, et al. (2003) Infect. Immunity 71:1961-1971; Xu, et al. (1998) J.Exp. Med. 187:787-794; Kropf, et al. (2002) Eur. J. Immunol.32:2450-2459; Swirski, et al. (2002) J. Immunol. 169:3499-3506; Sweet,et al. (2001) J. Immunol. 166:6633-6639; Walzl, et al. (2001) J. Exp.Med. 193:785-792.

IL-1 family members typically bind to a heterodimeric members of theIL-1 receptor family. It was shown that another known IL-1R familymember, SIGIRR (single Ig IL-1 receptor related protein), complexes withT1/ST2 to form the functional receptor complex for IL-33. SIGIRR wasoriginally found as an orphan IL-1R member (see, e.g., Garlanda, et al.(2004) Proc. Natl. Acad. Sci. 101:3522-3526; Clark, et al. (2003) GenomeRes. 13:2265-2270; Thomassen et al. (1999) Cytokine 11:389-399; GenBankAccession No. NP_068577; GenBank Accession No. NM_021805; GenBankAccession No. NP_075546; and GenBank Accession No. NM_0230459). SIGIRRis a widely expressed IL-1R member.

In precipitation experiments using biotinylated mature human IL-33(residues 112-270 of SEQ ID NO: 2), T1/ST2-Fc fusion, and SIGIRR-Fcfusion, it was shown that IL-33 could bind both receptor fusionproteins, however, the binding of IL-33 to SIGIRR was weaker as comparedto IL-33 and T1/ST2 binding. To test the signaling capabilities ofeither or both receptors, an NF-κB-dependent assay was run.Co-expression of both T1/ST2 and SIGIRR was both necessary andsufficient to activate NF-κB signaling and MAP kinase upon stimulationwith IL-33. Activation of JNK kinases was also observed.

III. Agonists, Antagonists, and Binding Compositions

The present invention provides agonists and antagonists of IL-33,including binding compositions that specifically bind to IL-33 or toIL-33 receptor complex (T1/ST2 and SIGIRR). Binding compositions includeantibodies, antibody fragments, and soluble receptors. The presentinvention contemplates blocking antibodies that bind to IL-33 or toIL-33R, or agonistic antibodies that stimulate signaling via the IL-33Rcomplex. The binding compositions of the present invention also includenucleic acids that specifically hybridize to nucleic acids encodingIL-33 or IL-33R, e.g., anti-sense nucleic acids and small interferenceRNA (siRNA) Anti-idiotypic antibodies may also be used. Human IL-33 isdisclosed by GenBank NM_033439. Regions of increased antigenicity,suitable for preparing anti-IL-33 antibodies, occur at, e.g., aminoacids 1-23; 30-38; 61-78; 84-93; 99-106; 127-133; 139-144; 148-158;166-180; 196-204; 231-237; and 252-257, of GenBank NM_033439, accordingto a Parker plot using Vector NTI® Suite (Informax, Inc, Bethesda, Md.).

Receptors based on these extracellular regions are not limited by theseexact N-terminal and C-terminal amino acids, but may be longer orshorter, e.g., by one, two, three, or more amino acids, as long as theligand binding properties are substantially maintained. Fusion proteinsbased on the soluble receptors are also contemplated, e.g., forfacilitating purification or stability or for providing a functionaldomain, e.g., a toxic polypeptide.

Monoclonal, polyclonal, and humanized antibodies can be prepared (see,e.g., Sheperd and Dean (eds.) (2000) Monoclonal Antibodies, Oxford Univ.Press, New York, N.Y.; Kontermann and Dubel (eds.) (2001) AntibodyEngineering, Springer-Verlag, New York; Harlow and Lane (1988)Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., pp. 139-243; Carpenter, et al. (2000) J.Immunol. 165:6205; He, et al. (1998) J. Immunol. 160:1029; Tang, et al.(1999) J. Biol. Chem. 274:27371-27378; Baca, et al. (1997) J. Biol.Chem. 272:10678-10684; Chothia, et al. (1989) Nature 342:877-883; Footeand Winter (1992) J. Mol. Biol. 224:487-499; U.S. Pat. No. 6,329,511issued to Vasquez, et al.). Muteins and variants of antibodies andsoluble receptors are contemplated, e.g., pegylation or mutagenesis toremove or replace deamidating Asn residues.

Purification of antigen is not necessary for the generation ofantibodies. Immunization can be performed by DNA vector immunization,see, e.g., Wang, et al. (1997) Virology 228:278-284. Alternatively,animals can be immunized with cells bearing the antigen of interest.Splenocytes can then be isolated from the immunized animals, and thesplenocytes can fused with a myeloma cell line to produce a hybridoma(Meyaard, et al. (1997) Immunity 7:283-290; Wright, et al. (2000)Immunity 13:233-242; Preston, et al. (1997) Eur. J. Immunol.27:1911-1918). Resultant hybridomas can be screened for production ofthe desired antibody by functional assays or biological assays, that is,assays not dependent on possession of the purified antigen. Immunizationwith cells may prove superior for antibody generation than immunizationwith purified antigen (Kaithamana, et al. (1999) J. Immunol.163:5157-5164).

Antibodies will usually bind with at least a K_(D) of about 10⁻³M, moreusually at least 10⁻⁶M, typically at least 10⁻⁷M, more typically atleast 10⁻⁸M, preferably at least about 10⁻⁹M, and more preferably atleast 10⁻¹⁰ M, and most preferably at least 10⁻¹¹M (see, e.g., Presta,et al. (2001) Thromb. Haemost. 85:379-389; Yang, et al. (2001) Crit.Rev. Oncol. Hematol. 38:17-23; Carnahan, et al. (2003) Clin. Cancer Res.(Suppl.) 9:3982s-3990s).

Soluble receptors comprising the extracellular domains of the IL-33receptor complex (T1/ST2 and SIGIRR) can be prepared, as thecytoplasmic, transmembrane, and extracellular regions of each of thesubunits have been identified (see, e.g., Lecart, et al. (2002) Eur. J.Immunol. 32:2979-2987; Mitcham, et al. (1996) J. Biol. Chem.271:5777-5783; and the Sequence Listing below).

Soluble receptors can be prepared and used according to standard methods(see, e.g., Jones, et al. (2002) Biochim. Biophys. Acta 1592:251-263;Prudhomme, et al. (2001) Expert Opinion Biol. Ther. 1:359-373;Fernandez-Botran (1999) Crit. Rev. Clin. Lab Sci. 36:165-224). Alsoprovided are compositions for siRNA interference (see, e.g., Arenz andSchepers (2003) Naturwissenschaften 90:345-359; Sazani and Kole (2003)J. Clin. Invest. 112:481-486; Pirollo, et al. (2003) Pharmacol.Therapeutics 99:55-77; Wang, et al. (2003) Antisense Nucl. Acid DrugDevel. 13:169-189).

IV. Therapeutic Compositions, Methods

The present invention provides methods for treating and diagnosinginnate response, asthma, allergies, and arthritis.

To prepare pharmaceutical or sterile compositions including an agonistor antagonist of IL-33, the reagent is mixed with a pharmaceuticallyacceptable carrier or excipient. Formulations of therapeutic anddiagnostic agents can be prepared by mixing with physiologicallyacceptable carriers, excipients, or stabilizers in the form of, e.g.,lyophilized powders, slurries, aqueous solutions, lotions, orsuspensions (see, e.g., Hardman, et al. (2001) Goodman and Gilman's ThePharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.;Gennaro (2000) Remington: The Science and Practice of Pharmacy,Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.)(1993) Pharmaceutical Dosage Forms: Parenteral Medications, MarcelDekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms:Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990)Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weinerand Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc.,New York, N.Y.).

Selecting an administration regimen for a therapeutic depends on severalfactors, including the serum or tissue turnover rate of the entity, thelevel of symptoms, the immunogenicity of the entity, and theaccessibility of the target cells in the biological matrix. Preferably,an administration regimen maximizes the amount of therapeutic deliveredto the patient consistent with an acceptable level of side effects.Accordingly, the amount of biologic delivered depends in part on theparticular entity and the severity of the condition being treated.Guidance in selecting appropriate doses of antibodies, cytokines, andsmall molecules are available (see, e.g., Wawrzynczak (1996) AntibodyTherapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991)Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York,N.Y.; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy inAutoimmune Diseases, Marcel Dekker, New York, N.Y.; Baert, et al. (2003)New Engl. J. Med. 348:601-608; Milgrom, et al. (1999) New Engl. J. Med.341:1966-1973; Slamon, et al. (2001) New Engl. J. Med. 344:783-792;Beniaminovitz, et al. (2000) New Engl. J. Med. 342:613-619; Ghosh, etal. (2003) New Engl. J. Med. 348:24-32; Lipsky, et al. (2000) New Engl.J. Med. 343:1594-1602).

Antibodies, antibody fragments, and cytokines can be provided bycontinuous infusion, or by doses at intervals of, e.g., one day, oneweek, or 1-7 times per week. Doses may be provided intravenously,subcutaneously, topically, orally, nasally, rectally, intramuscular,intracerebrally, or by inhalation. A preferred dose protocol is oneinvolving the maximal dose or dose frequency that avoids significantundesirable side effects. A total weekly dose is generally at least 0.05μg/kg body weight, more generally at least 0.2 μg/kg, most generally atleast 0.5 μg/kg, typically at least 1 μg/kg, more typically at least 10μg/kg, most typically at least 100 μg/kg, preferably at least 0.2 mg/kg,more preferably at least 1.0 mg/kg, most preferably at least 2.0 mg/kg,optimally at least 10 mg/kg, more optimally at least 25 mg/kg, and mostoptimally at least 50 mg/kg (see, e.g., Yang, et al. (2003) New Engl. J.Med. 349:427-434; Herold, et al. (2002) New Engl. J. Med. 346:1692-1698;Liu, et al. (1999) J. Neurol. Neurosurg. Psych. 67:451-456; Portielji,et al. (20003) Cancer Immunol. Immunother. 52:133-144). The desired doseof a small molecule therapeutic, e.g., a peptide mimetic, naturalproduct, or organic chemical, is about the same as for an antibody orpolypeptide, on a moles/kg body weight basis. The desired plasmaconcentration of a small molecule therapeutic is about the same as foran antibody, on a moles/kg body weight basis.

An effective amount for a particular patient may vary depending onfactors such as the condition being treated, the overall health of thepatient, the method route and dose of administration and the severity ofside affects (see, e.g., Maynard, et al. (1996) A Handbook of SOPs forGood Clinical Practice, Interpharm Press, Boca Raton, Fla.; Dent (2001)Good Laboratory and Good Clinical Practice, Urch Publ., London, UK).

Typical veterinary, experimental, or research subjects include monkeys,dogs, cats, rats, mice, rabbits, guinea pigs, horses, and humans.

Determination of the appropriate dose is made by the clinician, e.g.,using parameters or factors known or suspected in the art to affecttreatment or predicted to affect treatment. Generally, the dose beginswith an amount somewhat less than the optimum dose and it is increasedby small increments thereafter until the desired or optimum effect isachieved relative to any negative side effects. Important diagnosticmeasures include those of symptoms of, e.g., the inflammation or levelof inflammatory cytokines produced. Preferably, a biologic that will beused is derived from the same species as the animal targeted fortreatment, thereby minimizing a humoral response to the reagent.

Methods for co-administration or treatment with a second therapeuticagent, e.g., a cytokine, steroid, chemotherapeutic agent, antibiotic, orradiation, are well known in the art (see, e.g., Hardman, et al. (eds.)(2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics,10^(th) ed., McGraw-Hill, New York, N.Y.; Poole and Peterson (eds.)(2001) Pharmacotherapeutics for Advanced Practice: A Practical Approach,Lippincott, Williams & Wilkins, Phila., Pa.; Chabner and Longo (eds.)(2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams &Wilkins, Phila., Pa.). An effective amount of therapeutic will decreasethe symptoms typically by at least 10%; usually by at least 20%;preferably at least about 30%; more preferably at least 40%, and mostpreferably by at least 50%.

The route of administration is by, e.g., topical or cutaneousapplication, injection or infusion by intravenous, intraperitoneal,intracerebral, intramuscular, intraocular, intraarterial,intracerebrospinal, intralesional, or pulmonary routes, or by sustainedrelease systems or an implant (see, e.g., Sidman et al. (1983)Biopolymers 22:547-556; Langer, et al. (1981) J. Biomed. Mater. Res.15:167-277; Langer (1982) Chem. Tech. 12:98-105; Epstein, et al. (1985)Proc. Natl. Acad. Sci. USA 82:3688-3692; Hwang, et al. (1980) Proc.Natl. Acad. Sci. USA 77:4030-4034; U.S. Pat. Nos. 6,350,466 and6,316,024).

V. Kits and Diagnostic Reagents

Diagnostic methods for inflammatory disorders, e.g., psoriasis, Crohn'sdisease, rheumatoid arthritis, asthma or allergy, atherosclerosis, andcancers, based on antibodies, nucleic acid hybridization, and the PCRmethod, are available.

This invention provides polypeptides of IL-33, fragments thereof,nucleic acids of IL-33, and fragments thereof, in a diagnostic kit,e.g., for the diagnosis of viral disorders, including of influenza A,and viral disorders of the respiratory tract and of mucosal tissues.Also provided are binding compositions, including antibodies or antibodyfragments, for the detection of IL-33, and metabolites and breakdownproducts thereof. Typically, the kit will have a compartment containingeither a IL-33 polypeptide, or an antigenic fragment thereof, a bindingcomposition thereto, or a nucleic acid, such as a nucleic acid probe,primer, or molecular beacon (see, e.g., Rajendran, et al. (2003) NucleicAcids Res. 31:5700-5713; Cockerill (2003) Arch. Pathol. Lab. Med.127:1112-1120; Zammatteo, et al. (2002) Biotech. Annu. Rev. 8:85-101;Klein (2002) Trends Mol. Med. 8:257-260).

A method of diagnosis can comprise contacting a sample from a subject,e.g., a test subject, with a binding composition that specifically bindsto a polypeptide or nucleic acid of IL-33 or IL-33 receptor. The methodcan further comprise contacting a sample from a control subject, normalsubject, or normal tissue or fluid from the test subject, with thebinding composition. Moreover, the method can additionally comprisecomparing the specific binding of the composition to the test subjectwith the specific binding of the composition to the normal subject,control subject, or normal tissue or fluid from the test subject.Expression or activity of a test sample or test subject can be comparedwith that from a control sample or control subject. A control sample cancomprise, e.g., a sample of non-affected or non-inflamed tissue in apatient suffering from an immune disorder. Expression or activity from acontrol subject or control sample can be provided as a predeterminedvalue, e.g., acquired from a statistically appropriate group of controlsubjects.

The kit may comprise, e.g., a reagent and a compartment, a reagent andinstructions for use, or a reagent with a compartment and instructionsfor use. The reagent may comprise an agonist or antagonist of IL-33, oran antigenic fragment thereof, a binding composition, or a nucleic acidin a sense and/or anti-sense orientation. A kit for determining thebinding of a test compound, e.g., acquired from a biological sample orfrom a chemical library, can comprise a control compound, a labeledcompound, and a method for separating free labeled compound from boundlabeled compound. The control compound can comprise a segment of thepolypeptide of IL-33 or IL-33 receptor or a nucleic acid encoding IL-33or IL-33 receptor. The segment can comprise zero, one, two, or moreantigenic fragments.

A composition that is “labeled” is detectable, either directly orindirectly, by spectroscopic, photochemical, biochemical,immunochemical, isotopic, or chemical methods. For example, usefullabels include ³²P, ³³P, ³⁵S, ¹⁴C, ³H, ¹²⁵I, stable isotopes,fluorescent dyes, electron-dense reagents, substrates, epitope tags, orenzymes, e.g., as used in enzyme-linked immunoassays, or fluorettes(Rozinov and Nolan (1998) Chem. Biol. 5:713-728).

Diagnostic assays can be used with biological matrices such as livecells, cell extracts, cell lysates, fixed cells, cell cultures, bodilyfluids, or forensic samples. Conjugated antibodies useful for diagnosticor kit purposes, include antibodies coupled to dyes, isotopes, enzymes,and metals, see, e.g., Le Doussal, et al. (1991) New Engl. J. Med.146:169-175; Gibellini, et al. (1998) J. Immunol. 160:3891-3898; Hsingand Bishop (1999) New Engl. J. Med. 162:2804-2811; Everts, et al. (2002)New Engl. J. Med. 168:883-889. Various assay formats exist, such asradioimmunoassays (MA), ELISA, and lab on a chip (U.S. Pat. Nos.6,176,962 and 6,517,234).

Gene expression data is useful tool in the diagnosis and treatment ofdiseases and pathological conditions (see, e.g., Li and Wong (2001)Genome Informatics 12:3-13; Lockhart, et al. (1996) Nature Biotechnol.14:1675-1680; Homey, et al. (2000) J. Immunol. 164:3465-3470; Debets, etal. (2000) J. Immunol. 165:4950-4956).

VI. Uses

The present invention provides methods for the treatment and diagnosisof inflammatory and immune disorders, including inappropriate orineffective response to infection. Provided are methods relating to,e.g., asthma, allergies, arthritis, disorders involving eosinophilicinflammation, and disorders involving pathogenic or ineffective TH2-typeresponse.

The present invention provides methods for stimulating immune defenseagainst bacteria, parasites, and viruses, intracellular pathogens, andcancers and tumors. Provided are methods for the treatment ofintracellular bacteria. Intracellular bacterial species includeSalmonella sp., Shigella sp., Listeria sp., Francisella sp.,Mycobacteria sp. (tuberculosis; leprosy), Legionella sp., Rickettsiasp., Orienta sp., Ehrlichia sp., Anaplasma sp., Neorickettsia sp.,Chlamydia sp., and Coxiella sp. Additionally, IFNgamma mediates responseto parasites, e.g., Plasmodia sp. (malaria), Toxoplasma sp., Leishmaniasp., Trypanosoma sp., and Cryptosporidium sp. Provided are methods fortreating viruses, e.g., HIV, orthopoxviruses, such as variola virus andvaccinia virus (smallpox), and herpesviruses, includingalphaherpesviruses, e.g., Herpes Simplex virus, and betaherpesviruses,e.g., Cytomegalovirus. Also provided are methods for the treatment ofchronic inflammatory disorders (see, e.g., Kent, et al. (2000) Vaccine18:2250-2256; Ismail, et al. (2002) FEMS Microbiol. Lett. 207:111-120;Kaufmann (2001) Nature Revs. Immunol. 1:20-30; Goebel and Gross (2001)TRENDS Microbiol. 9:267-273; Heussler, et al. (2001) Int. J. Parasitol.31:1166-1176; Luder, et al. (2001) Carsten, et al. (2001) TRENDSParasitol. 17:480-486; Rook, et al. (2001) Eur. Resp. J. 17:537-557;Stenger and Rollinghoff (2001) Ann. Rheum. Dis. 60:iii43-iii46; Haas, etal. (2002) Am. J. Dermatopathol. 24:319-323; Dorman and Holland (2000)Cytokine Growth Factor Revs. 11:321-333; Smith, et al. (2002) J. Gen.Virol. 83 (Pt. 12) 2915-2931; Cohrs and Gilden (2001) Brain Pathol.11:465-474; Tannenbaum and Hamilton (2002) Sem. Cancer Biol. 10:113-123;Ikeda, et al. (2002) Cytokine Growth Factor Revs. 13:95-109; Klimp, etal. (2002) Crit. Rev. Oncol. Hematol. 44:143-161; Frucht, et al. (2001)TRENDS Immunol. 22:556-560).

The present invention provides methods of treating or diagnosing aproliferative condition or disorder, e.g., cancer of the uterus, cervix,breast, prostate, testes, penis, gastrointestinal tract, e.g.,esophagus, oropharynx, stomach, small or large intestines, colon, orrectum, kidney, renal cell, bladder, bone, bone marrow, skin, head orneck, skin, liver, gall bladder, heart, lung, pancreas, salivary gland,adrenal gland, thyroid, brain, ganglia, central nervous system (CNS) andperipheral nervous system (PNS), and immune system, e.g., spleen orthymus. The present invention provides methods of treating, e.g.,immunogenic tumors, non-immunogenetic tumors, dormant tumors,virus-induced cancers, e.g., epithelial cell cancers, endothelial cellcancers, squamous cell carcinomas, papillomavirus, adenocarcinomas,lymphomas, carcinomas, melanomas, leukemias, myelomas, sarcomas,teratocarcinomas, chemically-induced cancers, metastasis, andangiogenesis. The invention also contemplates reducing tolerance to atumor cell or cancer cell antigen, e.g., by modulating activity of aregulatory T cell (Treg) (see, e.g., Ramirez-Montagut, et al. (2003)Oncogene 22:3180-3187; Sawaya, et al. (2003) New Engl. J. Med.349:1501-1509; Farrar, et al. (1999) J. Immunol. 162:2842-2849; Le, etal. (2001) J. Immunol. 167:6765-6772; Cannistra and Niloff (1996) NewEngl. J. Med. 334:1030-1038; Osborne (1998) New Engl. J. Med.339:1609-1618; Lynch and Chapelle (2003) New Engl. J. Med. 348:919-932;Enzinger and Mayer (2003) New Engl. J. Med. 349:2241-2252; Forastiere,et al. (2001) New Engl. J. Med. 345:1890-1900; Izbicki, et al. (1997)New Engl. J. Med. 337:1188-1194; Holland, et al. (eds.) (1996) CancerMedicine Encyclopedia of Cancer, 4^(th) ed., Academic Press, San Diego,Calif.).

The present invention provides methods for treating a proliferativecondition, cancer, tumor, or precancerous condition such as a dysplasia,with an agonist or antagonist of IL-33, with at least one additionaltherapeutic or diagnostic agent. The at least one additional therapeuticor diagnostic agent can be, e.g., a cytokine or cytokine antagonist,such as interferon-alpha, or anti-epidermal growth factor receptor,doxorubicin, epirubicin, an anti-folate, e.g., methotrexate orfluoruracil, irinotecan, cyclophosphamide, radiotherapy, hormone oranti-hormone therapy, e.g., androgen, estrogen, anti-estrogen,flutamide, or diethylstilbestrol, surgery, tamoxifen, ifosfamide,mitolactol, an alkylating agent, e.g., melphalan or cis-platin,etoposide, vinorelbine, vinblastine, vindesine, a glucocorticoid, ahistamine receptor antagonist, an angiogenesis inhibitor, radiation, aradiation sensitizer, anthracycline, vinca alkaloid, taxane, e.g.,paclitaxel and docetaxel, a cell cycle inhibitor, e.g., acyclin-dependent kinase inhibitor, a monoclonal antibody, a complex ofmonoclonal antibody and toxin, a T cell adjuvant, bone marrowtransplant, or antigen presenting cells, e.g., dendritic cell therapy.Vaccines can be provided, e.g., as a soluble protein or as a nucleicacid encoding the protein (see, e.g., Le, et al., supra; Greco andZellefsky (eds.) (2000) Radiotherapy of Prostate Cancer, HarwoodAcademic, Amsterdam; Shapiro and Recht (2001) New Engl. J. Med.344:1997-2008; Hortobagyi (1998) New Engl. J. Med. 339:974-984; Catalona(1994) New Engl. J. Med. 331:996-1004; Naylor and Hadden (2003) Int.Immunopharmacol. 3:1205-1215; The Int. Adjuvant Lung Cancer TrialCollaborative Group (2004) New Engl. J. Med. 350:351-360; Slamon, et al.(2001) New Engl. J. Med. 344:783-792; Kudelka, et al. (1998) New Engl.J. Med. 338:991-992; van Netten, et al. (1996) New Engl. J. Med.334:920-921).

A number of biomarkers and methods for scoring inflammatory disorders,e.g., psoriasis, Crohn's disease, and rheumatoid arthritis are available(see, e.g., Bresnihan (2003) Arthritis Res. Ther. 5:271-278; Barnero andDelmas (2003) Curr. Opin. Rheumatol. 15:641-646; Gionchetti, et al.(2003) Dig. Dis. 21:157-167; Wiik (2002) Autoimmune Rev. 1:67-72;Sostegni, et al. (2003) Aliment Pharmacol. Ther. 17 (Suppl. 2):11-17).

Biomarkers and methods for scoring cancer are also described (see, e.g.,Alison (ed.) (2001) The Cancer Handbook, Grove's Dictionaries, Inc., St.Louis, Mo.; Oldham (ed.) (1998) Principles of Cancer Biotherapy, 3^(rd).ed., Kluwer Academic Publ., Hingham, M A; Thompson, et al. (eds.) (2001)Textbook of Melanoma, Martin Dunitz, Ltd., London, UK; Devita, et al.(eds.) (2001) Cancer: Principles and Practice of Oncology, 6^(th) ed.,Lippincott, Phila, Pa.; Holland, et al. (eds.) (2000) Holland-FreiCancer Medicine, BC Decker, Phila., Pa.; Garrett and Sell (eds.) (1995)Cellular Cancer Markers, Humana Press, Totowa, N.J.; MacKie (1996) SkinCancer, 2^(nd) ed., Mosby, St. Louis; Moertel (1994) New Engl. J. Med.330:1136-1142; Engleman (2003) Semin. Oncol. 30(3 Suppl. 8):23-29; Mohr,et al. (2003) Onkologie 26:227-233).

The broad scope of this invention is best understood with reference tothe following examples, which are not intended to limit the inventionsto the specific embodiments.

EXAMPLES I. General Methods

Standard methods in biochemistry and molecular biology are described(see, e.g., Maniatis, et al. (1982) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.;Sambrook and Russell (2001) Molecular Cloning, 3^(rd) ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.; Wu (1993) RecombinantDNA, Vol. 217, Academic Press, San Diego, Calif.). Standard methods alsoappear in Ausbel, et al. (2001) Current Protocols in Molecular Biology,Vols. 1-4, John Wiley and Sons, Inc. New York, N.Y., which describescloning in bacterial cells and DNA mutagenesis (Vol. 1), cloning inmammalian cells and yeast (Vol. 2), glycoconjugates and proteinexpression (Vol. 3), and bioinformatics (Vol. 4).

Methods for protein purification including immunoprecipitation,chromatography, electrophoresis, centrifugation, and crystallization aredescribed (Coligan, et al. (2000) Current Protocols in Protein Science,Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis,chemical modification, post-translational modification, production offusion proteins, glycosylation of proteins are described (see, e.g.,Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 2,John Wiley and Sons, Inc., New York; Ausubel, et al. (2001) CurrentProtocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY,N.Y., pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for LifeScience Research, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia Biotech(2001) BioDirectory, Piscataway, N.J., pp. 384-391). Methods for theproduction, purification, and fragmentation of polyclonal and monoclonalantibodies are described (Coligan, et al. (2001) Current Protocols inImmunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane(1999) Using Antibodies, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; Harlow and Lane, supra). Standard techniques forcharacterizing ligand/receptor interactions are available (see, e.g.,Coligan, et al. (2001) Current Protocols in Immunology, Vol. 4, JohnWiley, Inc., New York).

Methods for flow cytometry, including fluorescence activated cellsorting (FACS), are available (see, e.g., Owens, et al. (1994) FlowCytometry Principles for Clinical Laboratory Practice, John Wiley andSons, Hoboken, N.J.; Givan (2001) Flow Cytometry, 2^(nd) ed.;Wiley-Liss, Hoboken, N.J.; Shapiro (2003) Practical Flow Cytometry, JohnWiley and Sons, Hoboken, N.J.). Fluorescent reagents suitable formodifying nucleic acids, including nucleic acid primers and probes,polypeptides, and antibodies, for use, e.g., as diagnostic reagents, areavailable (see, e.g., Molecular Probes (2003) Catalogue, MolecularProbes, Inc., Eugene, Oreg.; Sigma-Aldrich (2003) Catalogue, St. Louis,Mo.).

Standard methods of histology of the immune system are described (see,e.g., Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology andPathology, Springer Verlag, New York, N.Y.; Hiatt, et al. (2000) ColorAtlas of Histology, Lippincott, Williams, and Wilkins, Phila, Pa.;Louis, et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, NewYork, N.Y.).

Methods for using animal models, e.g., knockout mice, and cell-basedassays for the testing, evaluation, and screening of diagnostic,therapeutic, and pharmaceutical agents are available (see, e.g., Car andEng (2001) Vet. Pathol. 38:20-30; Kenyon, et al. (2003) Toxicol. Appl.Pharmacol. 186:90-100; Deurloo, et al. (2001) Am. J. Respir. Cell Mol.Biol. 25:751-760; Zuberi, et al. (2000) J. Immunol. 164:2667-2673;Temelkovski, et al. (1998) Thorax 53:849-856; Horrocks, et al. (2003)Curr. Opin. Drug Discov. Devel. 6:570-575; Johnston, et al. (2002) DrugDiscov. Today 7:353-363).

Software packages and databases for determining, e.g., antigenicfragments, leader sequences, protein folding, functional domains,glycosylation sites, and sequence alignments, are available (see, e.g.,GenBank, Vector NTI® Suite (Informax, Inc, Bethesda, Md.); GCG WisconsinPackage (Accelrys, Inc., San Diego, Calif.); DeCypher® (TimeLogic Corp.,Crystal Bay, Nev.); Menne, et al. (2000) Bioinformatics 16: 741-742;Menne, et al. (2000) Bioinformatics Applications Note 16:741-742; Wren,et al. (2002) Comput. Methods Programs Biomed. 68:177-181; von Heijne(1983) Eur. J. Biochem. 133:17-21; von Heijne (1986) Nucleic Acids Res.14:4683-4690).

II. Interleukin-100

Similarity of the amino acid sequence of human IL-33 to other members ofthe IL-1 family is as follows. Similarity with IL-1Ra is 34% similarity;IL-1delta is 36%; IL-1F10 is 36%; IL-1zeta is 9%; IL-1F8 is 32%;IL-1epsilon is 40%; and IL-1F9 is 31%.

Interleukin-100 was discovered by using computational sequence analysesand identified as a new IL-1 family member by secondary structurecomparison with members of the IL-1 family, respectively IL-1beta andIL-18. IL-1 family members are highly inflammatory regulators of theimmune system, released in response to pathogenic challenges. Human andmouse homologs of IL-33 were identified as well as a rat and dog IL-33.Gene expression analysis showed that human IL-33 was expressed inepithelial cells, smooth muscle cells, and mesangial cells. Uponstimulation with IL-1β and TNF-α, human IL-33 mRNA levels are highlyinduced in primary normal human dermal and lung fibroblasts as well asin bronchial smooth muscle cells. Expression of IL-33 mRNA in psoriaticskin samples as well as in lung pulmonary alveolar proteinosis wassignificantly elevated.

Similar to IL-1 and IL-18, IL-33 has no signal peptide. In stead, IL-33is made and secreted as a large preproprotein that requires extensiveprocessing to release the mature, biologically active form. It is likelythat prepro IL-33 is processed by a caspase. We identified a caspasecleavage site in the protein sequence and showed that in vitrotranslated human IL-33 is cleaved by recombinant constitutively activecaspase-1. To investigate the putative biological role of IL-33,recombinant proteins were expressed and purified in E. coli. Therefore,the IL-33 gene was cloned into a pET3a bacterial expression vector. TheN-terminus of the recombinant protein was selected by comparing themature sequence of IL-33 to IL-1β and IL-18 so that the mature,biologically active protein would lack the pro-domain. Amino acid 112 ofthe full-length protein was selected as the N-terminal amino acid.Recombinant protein was expressed and purified from E. coli. In vivostudies were carried out. The intraperitoneal (IP) injection ofrecombinant human IL-33 into mice (C57BL/6J), with a dosage of either 5ug/day or 50 ug/day, led to severe eosinophilia and splenomegaly after 7days. Serum levels of several cytokines were tested on day 3 and day 7.An induction of IL-5 up to 10000 pg/ml in the 50 ug rhIL-33/day groupand up to 1000 pg/ml in the 5 ug rhIL-33/day group was observed on day3. The serum levels of IL-5 decreased to 1100 pg/ml in the 50 ugrhIL-33/day group and in the 5 ug rhIL-33/day group to 500 pg/ml. IL-13serum levels were also detectable at day 7 after treatment with either 5ug or 50 ug IL-33/day at 30 pg/ml and 100 pg/ml, respectively. No IL-5and IL-13 could be detected in the PBS-treated control group.Furthermore, no elevated levels for IFN-γ, TNF-α, IL-12, IL-10, IL-6,IL-4, IL-2 or MCP-1 could be detected in IL-33 treated mice or in thecontrol group.

After IP injection of 50 ug rhIL-33/day for 2 days liver lymphocyteswere harvested. Cells were plated into culture dishes with 2×10e6/ml ofculture medium and were stimulated with 50 ng/ml PMA and 1 uM ionomycinfor 4 hr. During the last 2 hr, Brefelding A, a secretion inhibitor, wasadded. Cells were surface stained for CD3 and NK1.1 and intracellularlystained either for IL-5 or IL-4 and analyzed by FACS. CD3/NK1.1 positiveliver lymphocytes cells derived from mice treated for 2 days with IL-33showed an accumulation of IL-5 and IL-4. These results suggest thatIL-33 activates NKT cells to secrete IL-4 and IL-5.

To test if IL-33 binds NKT cells, biotinylated IL-33 was used for abinding experiment. Human NKT cells, derived from PBMCs, were incubatedeither with Strepdavidin-PE or biotinylated rhIL-33 and Strepdavidin-PE.The stained cells were analyzed by FACS. Binding of rhIL-33 to human NKTcells was observed. This binding could be competed with unbiotinylatedrhIL-33.

IL-1 family members exert their biological response by interacting withcell surface receptors. We have identified the orphan IL-1 receptorST2/T1 as a cellular receptor for IL-33. FACS staining of a mouse mastcell line with a ST2/T1-specific monoclonal antibody showed that thismast cell line expresses the ST2/T1 receptor. This staining could bespecifically and dose-dependently competed by incubating the mast cellline with IL-33 protein.

NKT cells are essential for airway inflammation and the production ofIL-4 and IL-13 in allergen-induced airway-hyper-reactivity in mousemodels of asthma. The induction of IL-5 and IL-13 by IL-33 in NKT cellssuggests that IL-33 can play a role in the induction of these diseases.The generation of therapeutic antibodies neutralizing IL-33 may bebeneficial in the treatment of these diseases.

NKT cells have been implicated in many diseases. Identification of IL-33as a modulator of NKT cells suggest that IL-33 can affect otherdiseases, such as lupus, multiple sclerosis, malignancies, airwayinflammation and infectious diseases. The induction of Th2 cytokines byIL-33, such as IL-5 and IL-13, may help in the protection againstmicrobial infection or in the protection against tumors.

IL-1 family members typically bind to two different members of the IL-1receptor family to form a complete receptor complex. The identificationof IL-33 and ST2/T1 as one of the subunits that make up the receptor forIL-33, makes it possible to identify the second IL-33 receptor subunit.Identification of the complete IL-33 receptor will allow detailedidentification of the biological responses induced by IL-33.

Real time PCR analysis using Taqman revealed that IL-33 was expressed bya number of cells and tissues (Table 1). The present invention providesagonists and antagonists of IL-33 for the modulation of inflammatory andautoimmune disorders and conditions, e.g., psoriasis, asthma, allergies,and inflammatory bowel disease, e.g., gastric inflammation, ulcerativecolitis, Crohn's disease, celiac disease, and irritable bowel syndrome.

TABLE 1 Real time PCR analysis of IL-33 expression, relative toubiquitin (1.0). Human skin psoriasis vulgaris Human skin, normaladjacent Skin sample no. Expression Skin sample no. Expression PS-034757 PS-034 261 PS-037 731 PS-037 267 PS-028 443 PS-028 285 PS-025 446PS-025 261 PS-023 602 PS-023 235 Colon control 0.5 Colon Crohn's 87 no.4003197A Colon Crohn's 125 no. 9609C144 Colon Crohn's 114 no. 403242AExpression of cytokines with Nippostrongylus brasiliensis infection, asdetermined by real time PCR Taqman analysis. Cytokine tested sampleIL-25 IL-13 IL-4 IL-5 IL-33 Untreated stomach 0.86 0.03 0.51 0.01 9.43stomach 2 day 1.46 0.69 0.09 0.02 56.4 stomach 4 day 0.73 0.67 0.39 0.0980.32 stomach 8 day 1.04 3.30 0.33 0.44 84.9 stomach 11 days 0.27 1.221.77 0.01 8.86 stomach 16 days 0.35 0.38 1.31 0.12 3.43 Untreatedcontrol lung 0.29 0.01 0.57 0.66 56.8 Lung nippo 2 days 0.91 0.37 0.610.65 90.37 Lung nippo 4 days 0.59 13.34 4.65 3.78 356.49 Lung nippo 8days 0.36 55.88 8.78 1.14 48.76 Lung nippo 11 days 0.35 33.93 16.05 2.11116.79 Lung nippo 16 days 0.04 23.77 25.48 0.84 30.65

IL-33 induction by IL-1β plus TNF-α (8 hours) was compared with IL-33induction with medium alone. Induction was studied in the indicated celltype (Table 2).

TABLE 2 IL-33 induction. ND means not detectable. Numbers are relativeto ubiquitin (1.0). Cell IL-1β plus TNF-α Medium only NHDF 3250  50 NHEKND ND NHBE 25  25 PAEC 100 200 NHLF 25 ND BSMC 2025 350

In vitro translated human IL-33 was found to be cleaved by caspase-1.Without caspase treatment, analysis by SDS PAGE revealed a band at about32 kDa, corresponding to pro-human IL-33. Treatment with caspase-1 for 1hour at 37° resulted in two bands of about equal intensity, onecorresponding to pro-IL-33, and the other migrating at about 20-22 kDa(mature human IL-33). Treatment for 2 hours at 37° resulted in the sametwo bands, but with about two thirds of the protein migrating at about22 kDa. Similar studies demonstrated that in vitro translated humanIL-33 could also be cleaved by elastase or by cathepsin G, to speciesmigrating at about 20-22 kDa, whereas MMP-3 treatment did not result incleavage under the conditions used. Amino acid 112 of IL-33 is believedto be the position of cleavage, producing the mature IL-33, due tohomology with other IL-1 family members.

T1/ST2 was identified as at least one subunit of the receptor for humanIL-33. Expression of T1/ST2 was as follows (Table 3).

TABLE 3 Expression of T1/ST2 by human or mouse cells. Cell type Humancells Mouse cells TH1-type T cells −/+ −/+ TH2-type T cells −/+ +++ Mastcells +++ ++ monocytes/PBMC treated ++ not determined with LPS Dendriticcells ex BM (not determined) +

Human IL-33 was cloned in a pET3a vector, and expressed in E. coli. Thecloned protein began with amino acid 112, and was 158 amino acids long(18 kDa). IPTG was used to induced expression, and the protein was foundto be water-soluble. The expressed protein was purified using a A-columnand Sephadex gel filtration. The purified preparation was tested forendotoxin, where the results demonstrated about 0.023 EU per microgramprotein. Analysis by SDS PAGE using a non-reducing conditions revealedthat at least 95% of the protein migrated at a single molecular weightof 18 kDa.

IL-33 was injected intraperitoneally (i.p.) into B6/Balb/c mice. Threegroups of mice were used: (1) Injection with phosphate buffered saline(PBS); (2) hIL-33 (5 micrograms/day); and (3) hIL-33 (50micrograms/day). The protocol also involved injections (i.p.) for 3days, with sacrifice after three days of treatment, or injections (i.p.)for 7 days, with sacrifice after seven days of treatment. Blood, serum,blood smears, white blood cell differentials, histology, was performed.Thymus/spleen cells suspensions were analyzed by FACS analysis.

IL-33 treatment induces IL-5 and IL-13, as determined by measuring serumlevels of IL-5 and IL-13 (Table 4). The cytokines IL-4, IL-5, and IL-13were also measured in various organs with IL-33 administration. Theorgans tested were thymus, lung, spleen, and liver. These threecytokines were all found to be induced, as determined after 7 daystreatment with IL-33. Increases were found at both levels of IL-33 (5and 50 microgram IL-33). For example, in lung, IL-4, IL-5, and IL-13expression with saline treatment was about 1.0, or less. But with IL-33(50 micrograms), expression of IL-4 was 8.0; of IL-5 was 11.0; and ofIL-13 was 41.0. IL-33 treatment also provoked increases in serum IgE andIgA. With 7 days treatment, IgE levels were 30,000 ng/ml (PBS) and17,000 ng/ml (50 micrograms IL-33). With 7 days treatment, IgA levelswere 90 ng/ml (PBS) and 420 ng/ml (50 micrograms IL-33). IL-33 treatmentalso resulted in splenomegaly, where spleen mass in the PBS (control)treated mouse was about 80 mg, 5 micrograms with IL-33 for 7 days (150mg spleen), and 50 micrograms with IL-33 for 7 days (190 mg spleen).IL-33 treatment also produced extramedullary hematopoiesis in thespleen, and thymus hypoplasia (decrease in thymus size), and hypoplasiaof the cortex of the thymus.

TABLE 4 IL-33 treatment and white blood cell counts, platelet counts,and cytokine levels, at days 3 and 7. 5 micrograms IL- 50 micrograms IL-PBS 33/.day 33/day White blood cell counts (white blood cells/microliterblood) Day 3 12,000 12,000 14,000 Day 7 12,000 20,000 25,000 Platelets(platelets/microliter blood) Day 3 Day 7 1,200,000 1,000,000 600,000Neutrophils Day 3 500 350 520 Day 7 750 700 1200 Lymphocytes Day 310,000 10,000 7,500 Day 7 11,000 15,500 18,000 Monocytes Day 3 230 130200 Day 7 400 250 300 Eosinophils Day 3 420 220 130 Day 7 500 2000 1750Serum levels of IL-5 (pg/ml) Day 3 ND 500 7500 Day 7 ND 500 1100 Serumlevels of IL-7 (pg/ml) Day 7 ND 40 78

Transformed and non-transformed cells were injected into mice, followedby an incubation period of the cells within the mouse, and retrieval andpurification of the injected cells, with assessment of T1/ST2 expression(Table 5). Non-transformed mammary gland cells and ras-transformedmammary gland cells were used. With injection of the ras-transformedcells into a host immune-deficient nude mouse, the ras-transformed cellsexpressed T1/ST2 at increased levels, that is, expression was 103 (Table5). With injection of the ras-transformed cells into a host mouse havingan intact immune system, retrieval of the transformed cell, and Taqman®analysis, revealed greater increases in T1/ST2 expression. It isbelieved that these increases in T1/ST2 expression reflect increases insoluble T1/ST2, where the soluble T1/ST2 acts as a decoy. When solubleT1/ST2 acts as a decoy, it binds to IL-33, and inhibits the host frommounting a TH2-type immune response against the tumor cells. The hostmice with intact immune systems that were used, were Xtb mice and XBalbmice (Table 6). The soluble version of T1/ST2 (also known as Fit 1) isdescribed (see, e.g., Bergers, et al. (1994) EMBO J. 13:1176-1188;Reikerstorfer, et al. (1995) J. Biol. Chem. 270:17645-17648).

IL-33 was administered to mice harboring 4T1 breast cancer. AdministeredIL-33 was effective in reducing tumor size (Table 6). The presentinvention provides agonists of IL-33, e.g., IL-33 or a nucleic acidencoding IL-33, for the treatment of proliferative conditions, includingcancers and tumors.

TABLE 5 Injection of ras-transformed cells into nude mice andimmunocompetant mice, followed by Taqman analysis of T1/ST2 expressionby recovered ras-transformed cells. Expression of T1/ST2 Nude mouse host103 Xtb mouse host 421 XBalb mouse host 669

TABLE 6 Administered IL-33 reduces tumor size in mice. Tumor size (mm³)Treatment 23 24 25 26 27 28 29 30 31 32 33 IL-33 175 125 125 200 180 170205 200 200 240 240 no IL-33 175 200 225 240 280 300 370 405 430 470 500

Taqman analysis of human tissue samples revealed decreased expression ofIL-33 in various cancers, e.g., breast cancer and ovarian cancer (Table7). The results indicate that the tumor cells refrain from producingIL-33, in order to avoid activating the immune system to mount ananti-tumor response (Table 7).

TABLE 7 Real time PCR analysis of IL-33 expression by human cancertissues. Cancerous tissue Normal adjacent tissue Human breast 6221 30.4435.0 (infiltrating duct) Human breast 6612 59.7 54.7 (infiltratingduct) Human breast 6652 2.4 292.0 (infiltrating duct) Human breast7748/02 2.6 411.0 lobular Human breast 7156 lobular 77.4 189.1 Humanovary papillary 1872.7 609.4 serous cystadenocarcinoma 1590 Human ovarypapillary 206.6 793.9 serous cystadenocarcinoma 3572/02 Human ovarypapillary 235.1 1052.7 serous cystadenocarcinoma 246869

IL-33 treatment prolonged experimental autoimmune encephalitis, ananimal model for multiple sclerosis. EAE was induced by proteolipidprotein (PLP) (Kjellen, et al. (2001) J. Neuroimmunol. 120:25-33; Laman,et al. (2001) J. Neuroimmunol. 119:124-130; Fife, et al. (2001) J.Immunol. 166:7617-7624). Three groups of mice were used for injectionsof PBS, 0.5 micrograms IL-33, or 2.0 micrograms of IL-33, with dailyi.p. injections from day 0 to day 12. Disease scores were assessed ondays 7 to 23 (Table 8). The results demonstrated that in PBS-treatedmice, the disease spontaneously resolved. However, with treatment witheither dose of IL-33, the disease was prolonged, higher than that foundwith PBS treatment, and maintained itself at a disease score of between2.5-3.0 (Table 8). The present invention provides an antagonist of IL-33for the treatment of autoimmune disorders, including autoimmunedisorders of the central nervous system, e.g., multiple sclerosis.

TABLE 8 EAE disease score in mice at selected days after treatment withPBS or with IL-33. Day 11 Day 13 Day 15 Day 17 Day 19 Day 21 PBS 0 2.051.75 0.45 0.20 0.0 0.5 micrograms 0 0.75 1.15 2.4 2.9 2.9 IL-33 2.0micrograms 0 1.95 1.95 3.1 2.3 2.55 IL-33

IL-33 treatment led to IL-5 induction in NKT cells. NKT cells wereidentified by the presence of both the NK1.1 marker and the CD3 marker.Black-6 mice were treated for two days with PBS or 50 micrograms/day ofIL-33. Liver lymphocytes were isolated, and restimulated with PMAionomycin for 3 hours and brefeldin for 1 hour. The results demonstratedthat IL-33 induced IL-5 in NKT cells.

Anti-T1/ST2 antibody was tested for its ability to bind wild type mastcells (WTMC), and the influence of added IL-33 on binding of thisantibody to the mast cells. Adding IL-33 abolished the ability ofanti-T1/ST2 antibody to bind the mast cells, demonstrating that thereceptor of IL-33 is T1/ST2.

III. In Vivo Effects of IL-33 Antibody Treatment

A mouse monoclonal antibody against human IL-33 was raised using methodswell known in the art (see above). To test the ability of this antibodyto antagonize IL-33 activity, Balb/c mice were injected subcutaneouslywith 0.2 mg of anti-IL-33 antibody on day 0. On day 1, the mice wereinjected intraperitonally with 100 ng of mIL-33. Serum was collected onday 2, and IL-5 levels measured. Treatment with the anti-IL-33 antibodyresulted in little to no production of IL-5 when compared to micetreated with IL-33 alone and mice treated with isotype control antibodyand IL-33 (See FIG. 1).

IV. Treatment of Collagen Induced Arthritis (CIA)

B10.RIII mice, known to be susceptible to developing CIA, were injectedwith bovine collagen type II (bovine CII; Sigma) in complete Freund'sadjuvant (Difco). Mice were injected above the tail base with 100 ul ofa 1 mg/ml emulsion of bovine CII. A second boost dose was administeredat day 21. Mice were assessed by the following clinical scale: 0=normal;1=redness and/or swelling at one joint/site; 2=redness and/or swellingat more than on joint/site; and 3=redness and or swelling in the entirepaw. CIA induced mice have a percent disease onset of 70-90%.

Mice were treated at day 23 with 1 mg of anti-IL-33 antibody or isotypecontrol antibody. Antibodies were administered every 7 days for two moretreatments. Anti-IL-33 treated mice showed decreased disease scores aswell as a lower percent incidence of disease onset (see FIGS. 2 and 3).The anti-IL-33 treated mice also had a lower mean number of arthriticpaws (see FIG. 4).

V. Treatment of Experimental Autoimmune Encephilitis (EAE)

C57BL/6 mice were immunized with 50 ug of myelin oligodendrocyteglycoprotein (MOG) peptide to induce EAE. The clinical assessment of thedisease is scored as follows: 1=limp tail; 2=hind limb weakness;3=inability to right+single hind limb weakness; 4=inability toright+single hind limb paralysis; 5=bilateral hind limb paralysis;6=bilateral hind limb paralysis+abdomen collapse; and 7=6+moribund. EAEmice were treated subcutaneously with either 100 mg of anti-IL-33antibody or isotype control antibody. Anti-IL-33 treated mice showedlower disease scores and lower disease incidence than the control group(see FIGS. 5 and 6).

VI. Pull-Down Assay to Identify IL-33R Complex

IL-33 was biotinylated with EX-LINK Supho-NHS-Biotin (Pierce). Pull-downof 2 μg biotinylated IL-33 was performed in 500 μl RIPA-Lysis buffer(upstate cell singaling solution) with 50 μl of a 50% Slurry of Agarosebound Avidin D (Vector Laborities). 5 μg of either recombinantextra-cellular ST2-Fc (R&D Systems) or SIGIRR-Fc (R&D Systems) was used.After incubation overnight at 4° C. precipitates were washed 3× with 500μl RIPA-Lysis buffer. The precipitated proteins were separated bySDS-Page, electroblotted, and visualized by Western blot/ECL reactionwith antibodies specific against ST2 (R&D Systems) or SIGIRR (R&DSystems). Pull-down of biotinylated IL-33 with ST2-Fc or SIGIRR-Fc wasperformed in the same manner as above only Protein G-Sepharose (AmershamBioscience) was used instead of Agarose bound Avidin D. IL-33 presentswas visualized via a Streptavidin-HRP conjugate (Pierce) and ECLreaction.

VII. Phosphorylation of NF-κB and MAP Kinases

The mastcell line WTMC was described previously (see, e.g., Wright, etal. (2003). J. Immunol. 171:3034-3046.). Cells were lysed in RIPA lysisBuffer (Upstate) containing Complete Mini protease inhibitor cocktail(Roche) and 10 mM Na₃VO₄. Proteins were separated by SDS-Page,transferred to Immobilon-P membranes (Millipore) and immunoblotted usingantibodies to phosphorylated p65 NF-κB, p65 NF-κB, phosphorylated p44/42MAP kinases, p44/42 MAP kinases, phosphorylated p38 MAP kinase and p38MAP kinase (all Antibodies form Cell Signaling Technology).

VIII. Transient Transfection and Reporter Gene Assays

HEK293FT cells were seeded before transfection with an NF-κB-driven GFPreporter gene construct (pNF-κB-hrGFP; Stratagene) and with acombination of plasmids encoding for ST2, or SIGIRR or both, asindicated with Fugene-6 (Roche) according to manufacturer'srecommendations. Cells were split 24 hours after transfection. After 24hours cells were either left untreated or stimulated with mouse IL-33 atthe concentration of 50 ng/ml. Sixteen hours after stimulation, cellswere analyzed for GFP-expression by FACS.

TABLE 9 Sequence Identifiers SEQ ID NO: 1 Human IL-33 nucleic acidsequence SEQ ID NO: 2 Human IL-33 polypeptide sequence SEQ ID NO: 3Mouse IL-33 nucleic acid sequence SEQ ID NO: 4 Mouse IL-33 nucleic acidsequence SEQ ID NO: 5 Human T1/ST2 nucleic acid sequence SEQ ID NO: 6Human T1/ST2 polypeptide sequence SEQ ID NO: 7 Mouse T1/ST2 nucleic acidsequence SEQ ID NO: 8 Mouse T1/ST2 polypeptide sequence SEQ ID NO: 9Human SIGIRR nucleic acid sequence SEQ ID NO: 10 Human SIGIRRpolypeptide sequence SEQ ID NO: 11 Mouse SIGIRR nucleic acid sequenceSEQ ID NO: 12 Mouse SIGIRR polypeptide sequence

All citations herein are incorporated herein by reference to the sameextent as if each individual publication, patent application, or patentwas specifically and individually indicated to be incorporated byreference including all figures and drawings.

Many modifications and variations of this invention, as will be apparentto one of ordinary skill in the art can be made to adapt to a particularsituation, material, composition of matter, process, process step orsteps, to preserve the objective, spirit and scope of the invention. Allsuch modifications are intended to be within the scope of the claimsappended hereto without departing from the spirit and scope of theinvention. The specific embodiments described herein are offered by wayof example only, and the invention is to be limited by the terms of theappended claims, along with the full scope of equivalents to which suchclaims are entitled; and the invention is not to be limited by thespecific embodiments that have been presented herein by way of example.

What is claimed is:
 1. A method of modulating an immune disorder orcondition, comprising administering an effective amount of an agonist orantagonist of IL-33 of IL-33 Receptor complex (IL-33R).
 2. The method ofclaim 1, wherein the disorder or condition comprises: a) innateresponse; b) asthma or allergy; c) multiple sclerosis; d) aninflammatory bowel disorder; e) arthritis; f) infection; g) a cancer ortumor.
 3. The method of claim 2, wherein the infection comprises: a) anintracellular pathogen; b) a bacterium; c) a parasite; or d) a virus. 4.The method of claim 3, wherein the intracellular pathogen is: a)Leishmania sp.; b) Mycobacterium sp.; c) Listeria sp.; d) Toxoplasmasp.; e) Schistosoma; or f) a respiratory virus;
 5. The method of claim1, wherein the immune disorder or conditions comprises: a) TH1-typeresponse; or b) TH2-type response.
 6. The method of claim 5, wherein theTH2-type response comprises an early event in TH2-type response.
 7. Themethod of claim 1, wherein the arthritis comprises: a) rheumatoidarthritis; b) osteoarthritis; or c) psoriatic arthritis.
 8. The methodof claim 1, wherein the agonist comprises: a) IL-33 or; b) a nucleicacid.
 9. The method of claim 8, wherein the nucleic acid encodes IL-33.10. The method of claim 1, wherein the antagonist comprises a bindingcomposition from an antibody that specifically binds: a) IL-33; b) anIL-33R complex; or c) a complex of IL-33 and IL-33R.
 11. The method ofclaim 10, wherein the binding composition from an antibody comprises: a)a polyclonal antibody; b) a monoclonal antibody; c) a humanizedantibody, or a fragment thereof; d) an Fab, Fv, or F(ab′)₂ fragment; e)a peptide mimetic of an antibody; or f) a detectable label.
 12. Themethod of claim 1, wherein the antagonist comprises: a) a solubleIL-33R; b) a small molecule; or c) a nucleic acid.
 13. The method ofclaim 12, wherein the nucleic acid specifically hybridizes with apolynucleotide encoding IL-33.
 14. The method of claim 13, wherein thenucleic acid comprises: a) anti-sense nucleic acid; or b) smallinterference RNA (siRNA).
 15. A method of modulating blood cell countscomprising administering an effective amount of an agonist or antagonistof IL-33.
 16. The method of claim 15, wherein the IL-33 agonistincreases the counts of: a) total white blood cells; b) neutrophils; c)lymphocytes; or d) eosinophils.
 17. The method of claim 15, wherein theIL-33 antagonist increases the count of platelets.
 18. The method ofclaim 16, wherein the IL-33 antagonist decreases the counts of: a) totalwhite blood cells; b) neutrophils; c) lymphocytes; or d) eosinophils.19. A method of diagnosing the immune condition or disorder of claim 1,comprising contacting a binding composition to a biological sample,wherein the binding composition specifically binds to IL-33, andmeasuring or determining the specific binding of the binding compositionto the biological sample.
 20. A kit for the diagnosis of the immunecondition or disorder of claim 1, comprising a compartment and a bindingcomposition that specifically binds to: a) IL-33; b) an IL-33R complex;c) a complex of IL-33 and IL-33R; or d) or a nucleic acid encodingIL-33.